MicroRNAs (miRNAs) are endogenous noncoding single-stranded small RNAs. Numerous studies have shown that miRNAs have pivotal roles in the occurrence and development of myocardial fibrosis (MF). However, miRNA expression profile in rats with MF after myocardial infarction (MI) is not well understood. The present study aimed to find the potential miRNA for MF post MI.SPF male Sprague-Dawley (SD) rat models of acute myocardial infarction (AMI) were established by ligating the anterior descending branch of the left coronary artery, while sham-operated rats were only threaded without ligation as a control group. Hematoxylin-eosin and Masson trichrome staining were used to detect myocardial histopathological changes for model evaluation. The differentially expressed miRNAs were detected by using the Agilent Rat miRNA gene chip in the myocardial tissue of the infarct marginal zone. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed by DAVID. The expression of miR-199a-5p was verified by real-time fluorescence quantitative PCR (qRT-PCR). Transfected miR-199a-5p mimics into cardiac fibroblasts (CFs) to construct cell models of miR-199a-5p overexpression. Dual-luciferase reporter assay was employed to validate the target gene of miR-199a-5p. The protein expression of the target gene in CFs transfected with miR-199a-5p mimics were detected by Western blot.Myocardial fibrosis was exacerbated in the model group compared with the control group. Thirteen differentially expressed miRNAs between the two groups were screened and their expression levels in the model group were all higher than those in the control group. The expression of miR-199a-5p was significantly increased in the model group in qRT-PCR, which was consistent with the results of the gene chip. KEGG enrichment analysis showed that the target genes of miR-199a-5p were enriched in the insulin signaling pathway. Furthermore, dual-luciferase reporter assay indicated that miR-199a-5p could negatively regulate the expression of GSK-3β. After transfection, the expression of miR-199a-5p was increased in the miR-199a-5p mimics group. The protein expression of GSK-3β was decreased in CFs transfected with miR-199a-5p mimics.Our study identified miR-199a-5p could promote the progression of myocardial fibrosis after myocardial infarction by targeting GSK-3β, which provides novel targets for diagnosis and treatment of MF. In recent years, a national survey of 1.7 million Chinese adults reported that approximately one in 10 adults had a high risk of cardiovascular disease (CVD) . MyocardMicroRNAs (miRNAs) are a class of highly conserved endogenous noncoding single-stranded small RNAs with a length of 18–22 nucleotides. They are involved in regulation at the posttranscriptional and translational levels through the degradation and translation inhibition of messenger RNA (mRNA). It is estimated that at least 60% of all mammalian genes may be regulated by miRNAs . MiRNAs A growing number of studies have shown that energy metabolism imbalances such as glucose fluctuation can exacerbate cardiac remodeling and MF . Study hPrevious studies have found that MI in rats can cause the accumulation of collagen-based ECM, and lead to MF, however, the potential targets are not fully understood. With the development of the microarray technology and bioinformatics techniques, the disease-related miRNAs have been discovered. Therefore, in this study , rat modad libitum food and water. All protocols and applications of animals in this study were approved by The Animal Care & Welfare Committee of Dongzhimen Hospital, Beijing University of Chinese Medicine (No: 16–25). The rats were divided into model group and sham-operated group by random number table. A model of AMI was established by ligating the left anterior descending coronary artery (LAD) of the rat heart. The rat was anesthetized intraperitoneally with 1% sodium pentobarbital (40 mg/kg). The skin was cut laterally at the third and fourth ribs of the left chest, approximately 1.5 cm in length. The musculature was bluntly separated, the rib was braced with a thoracotomy and the thymus was fixed upward to fully enlarge the surgical field, and the pericardium was carefully torn open to expose the ligation site of the rat heart. The anterior descending branch of the left coronary artery was ligated with a 5/0 line approximately 2 mm below the left atrial appendage and the arterial cone. The anterior myocardial wall was quickly ischemic and whitening, and the electrocardiogram (ECG) revealed saddle-back-like ST-segment elevations. After 24 h, the ECG was conducted again, and pathological Q waves appeared in the chest leads and lead I and aVL, indicating that the model was successful  rats weighing 200 ± 20 g were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd., Raised in the Barrier Animal Laboratory of Dongzhimen Hospital, Beijing University of Chinese Medicine, with a 12 h light/dark cycle and ccessful . The shaDehydrated, paraffin-embedded myocardial tissues were cut into 3-μm-thick sections. The sections were dewaxed in xylene and rehydrated stepwise in a descending ethanol series. For hematoxylin-eosin (HE) staining, the sections were stained with hematoxylin staining solution and eosin staining solution . For Masson trichrome staining, the sections were dyed with nuclear staining, cytoplasmic staining, color separation solution, and counterstaining solution  in sequence. The samples were dehydrated with ethanol in a series of concentrations and cleared with xylene. Coverslips were mounted with neutral balsam and sections were photographed by light microscopy. With the help of Image-Pro Plus 6.0, the area of myocardial collagen could be quantified. Collagen volume fraction (CVF) = Collagen area/ × 100%. Five nonoverlapping fields were randomly selected for each section (×200), and the results are presented as the mean.p value  based on a t test were used to screen differentially expressed miRNAs. The differentially expressed miRNAs were clustered in an unsupervised hierarchy, and the expression patterns of the differentially expressed miRNAs among different samples were displayed in the form of heatmaps. The microarray data discussed in this study have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) and are accessible through (GEO) Series accession number GSE208159.Due to the cost of microarray, three myocardial tissue samples were randomly selected from each group for microarray detection. Total RNA was extracted from myocardial tissue according to the mirVanaTM RNA Isolation Kit . The Agilent Rat miRNA gene chip was used in this research. The hybridization and scanning of the miRNA gene chip were carried out. The standardized data were filtered, and at least one set of 100% probes marked as “Detected” in the two sets of samples used for comparison was left for subsequent analysis. The fold change value and https://david.ncifcrf.gov/) is a common software for target genes. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to perform enrichment analyses to elucidate biological functions and pathways regulated by miRNAs (P < 0.05). The protein-protein interaction (PPI) network was constructed using the STRING database and Cytoscape software.TargetScan and microRNAorg databases were used to predict target genes of differentially expressed miRNAs, and the intersection was taken for subsequent analysis. Database for Annotation, Visualization and Integrated Discovery (DAVID) . The RNA to be tested was reverse transcribed into cDNA using the miScript II Reverse Transcription Kit . Using cDNA as a template, real-time PCR was performed with QuantiFast® SYBR® Green PCR Master Mix . Reactions were incubated at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The expression levels of microRNAs were normalized to U6 and calculated using the 22. After 90 min, the unattached cells were discarded, and the medium was replaced with 10% FBS-DMEM. The experiments used 3–5 generations of cardiac fibroblasts (CFs).The 3-day-old Wistar suckling rats were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. All protocols and applications of animals in this study were approved by The Animal Care & Welfare Committee of Dongzhimen Hospital, Beijing University of Chinese Medicine (No: 21–06). The 3-day-old Wistar suckling rats were sacrifice by cervical dislocation and disinfected with alcohol three times. The skin on the chest was exposed, and cut perpendicular to the ribs along the left margin of the breastbone of the infant rats at 0.5 cm with sterile ophthalmic straight scissors. The heart was extruded by gently squeezing the incision, and the apex of heart was cut off with a sterile ophthalmic curved scissors and quickly placed in precooled PBS . Using the Neonatal Heart Dissociation Kit , the myocardial tissue was digested under aseptic conditions. The digested tissue was seeded in culture flasks with DMEM  containing 15% fetal bovine serum (FBS) , 1% penicillin, and streptomycin in an incubator at 37 °C and 5% CO4 CFs per well. After 24 h incubation at 37 °C and 5% CO2 incubator, CFs were sequentially fixed with 4% paraformaldehyde, permeated with 0.5% triton and inactivated endogenous peroxidase with 3% H2O2. CFs were incubated with anti-Vimentin  and anti-alpha-smooth muscle actin (α-SMA)  antibodies overnight at 4 °C. After washing with PBS, the CFs were incubated with a secondary antibody. DAB chromogenic reagent was added in the dark environment, claybank was positive expression, and no chromogenic was negative expression. Hematoxylin restained the nucleus.CFs were inoculated on 24-well polylysine-coated sterile slides with 8 × 102 for 6 h and then the medium was replaced with 10% FBS-DMEM.CFs were digested and inoculated into culture plates. After 24 h, DMEM without FBS, growth factor, penicillin, and streptomycin was replaced and transfected with miR-199a-5p mimics . The CFs were cultured in an incubator at 37 °C and 5% CO293T cells were co-transfected with rno-miR-199a-5p mimic or miR-NC and pMIR-REPORT Luciferase-GSK3β 3′UTR (WT) or pMIR-REPORT luciferase-GSK3β3′UTR (MUT), respectively. A total of 48 h after transfection, Luciferase activity was detected by microplate reader.CFs was homogenized with RIPA lysis buffer containing protease and phosphatase inhibitors. After SDS–PAGE, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk powder. The PVDF membranes were incubated with anti-GSK-3β  and anti-GAPDH  antibodies overnight at 4 °C. After washing with Tris-buffered saline with 0.05% Tween-20 (TBST), the membranes were incubated with a secondary antibody. Membranes were placed in an enhanced chemiluminescence (ECL) mixture and then exposed, developed, and fixed in a dark room. Using GAPDH as the internal reference, the immunoreactive bands were analyzed using ImageJ analysis software.P < 0.05.The data are presented as the mean ± standard deviation (SD). Statistical analysis was performed with SPSS 20.0 and GraphPad Prism 8. When the data were normally distributed, an unpaired t test was conducted for comparisons between two groups. The nonparametric Kruskal–Wallis test was used when the data did not obey a normal distribution. Statistical significance was defined when P < 0.0001, t = 28.21, df = 8, HE staining showed that the structure of cardiomyocytes in the control group was complete, with uniform morphology, clear nuclei, a uniformly stained cytoplasm, and tightly arranged muscle bundles. However, in the model group, the myocardial tissue structure was destroyed, with a large area of cardiomyocyte fragmentation, nuclear pyrosis and deformation, disordered arrangement of muscle fibers, and widening of space . To inveFC > 2, P < 0.05), and their expression levels in the model group were higher than those in the control group. In the longitudinal sample clustering tree, the model group was clustered into one cluster, while the control group was clustered into another cluster, indicating that the expression levels of miRNAs between the two groups had prominent differences. In the horizontal sample clustering tree, miRNAs with similar biological functions were clustered. Red indicates that the expression of miRNA was upregulated, and blue indicates that the expression of miRNA was downregulated; the brighter the color, the greater the difference.As shown in P < 0.01). KEGG enrichment analysis of common target genes was performed to predict the signaling pathways that differentially expressed miRNAs might target. As shown in P < 0.05).MiRNAs, small single-stranded noncoding RNAs (ncRNAs), can regulate the expression of target genes. In this study, two databases, microRNAorg and TargetScan, were used to predict target genes of the thirteen differentially expressed miRNAs, and 2155 common target genes were obtained . ThroughP = 0.01, t = 5.033, df = 4).Based on the miRNA expression profile, we selected miR-199a-5p that showed significantly upregulated expression for qRT-PCR validation in the myocardial tissue with MF post MI. As shown in CFs were extracted and identified by immunocytochemistry. Vimentin is commonly used to characterize the cells of fibroblast origin, while α-SMA is used to recognize myofibroblasts . ImmunohP < 0.0001, t = 13.22, df = 8, To further confirm the role of miR-199a-5p in MF, miR-199a-5p mimics were transfected into CFs. After transfection, the expression level of miR-199a-5p was increased in the miR-199a-5p mimics group, indicating that the transfections were successful (P < 0.01). KEGG analysis of miR-199a-5p target genes showed that a total of 7 signaling pathways . By Dual-luciferase reporter assay, we indicated that overexpression of rno-miR-199a-5p inhibited the luciferase activity of wild-type GSK-3β 3′-UTR, but it also suppress luciferase activity of mutated one (P = 0.0001, t = 15.01, df = 4 or P = 0.0013, t = 8.03, df = 4, P < 0.0001, t = 28.72, df = 4, Above results indicated that miR-199a-5p could regulate multiple functions and signaling pathways through multiple target genes and suggested that miR-199a-5p might be involved in the process of MF after MI through the insulin signaling pathway. Although there are many target genes of miR-199a-5p, KEGG analysis showed that only eight target genes of miR-199a-5p were clustered in the insulin signaling pathway, including Ppp1cb, Gsk3β, Slc2a4, Prkag2, Rps6kb1, Rhoq, Crkl, and Eif4e. We performed PPI analysis on these target genes, the results showed that GSK-3β interacted with multiple proteins, such as Slc2a4, Rps6kb1, and Eif4e, which was of great significance in the insulin signaling pathway , Fig. 6AA report from the American Heart Association updated in 2022 showed that in 2019, 27% of the world’s deaths were due to CVD, making it the leading cause of death globally, furthermore, in 2020, 19.05 million deaths were estimated for CVD globally, an increase of 18.71% compared to 2010 . In CVD,in vivo and in vitro (To find the potential miRNAs involved in MF after MI, the Agilent Rat miRNA gene chip was used to screen the differentially expressed miRNAs between the two groups. Results showed that miR-199a-5p was up-regulated in the model group, which was verified by qRT-PCR. A prior study indicated that members of the miR-199a family, namely, miR-199a-3p and miR-199a-5p, are potential regulators of cardiac homeostasis . The expin vitro . Therefoin vitro . Furtherin vitro . Target in vitro . GSK-3β in vitro .MiRNAs have been reported to regulate CVD through various mechanisms, such as the TGF-β signaling pathway  and the In summary, a novel miRNA, miR-199a-5p, was confirmed to be involved in the pathological process of MF post MI. However, the present study has several limitations. First of all, in this experiment, SD rats were used for surgery procedures, and Wistar suckling rats were used for fibroblast isolation. The design of this study was not rigorous enough because the same strain was not selected for the experiment. Currently, Wistar rats and SD rats are commonly used closed group rats. Although SD rats are bred from Wistar rats, they are not identical, which will be noted in future experiments. But it is worth mentioning that both of them can be used to prepare MF models. Secondly, there was left unblinded due to the nature of the analysis being carried out. Finally, due to the small amount of myocardial tissue in the control group and model group in this experiment, it was not enough for Western blot test to detect the expression of GSK-3β protein. In the future, we will build more animal models and reserve enough myocardial tissue for Western blot detection to show the expression of GSK-3β protein. Furthermore, multiple miRNAs and a complex network of signaling pathways are involved in the occurrence and development of MF. The pathogenesis is complex, and more elaborate mechanisms need to be elucidated in future studies.In conclusion, our study identified miR-199a-5p could promote the progression of myocardial fibrosis after myocardial infarction by targeting GSK-3β, which plays an important role in the insulin signaling pathway, which provides novel targets for diagnosis and treatment of MF.10.7717/peerj.14851/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14851/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.14851/supp-3Supplemental Information 3Click here for additional data file.


We found that N. brasiliensis infection and its derived proteins had suitable protective effects both in the initial and progressive stages of atherosclerosis, effectively reducing aortic arch plaque areas and liver lipid contents and downregulating serum LDL levels, which may be associated with the significant upregulation of serum anti-inflammatory cytokines (IL-10 and IL-4) and the down-regulation of proinflammatory cytokines (TNF-α and IFN-γ) in the serum. In conclusion, these data highlighted the effective regulatory role of N. brasiliensis and its derived proteins in the development and progression of atherosclerosis. This could provide a promising new avenue for the prevention and treatment of atherosclerosis.Atherosclerosis (AS) is a dominant and growing cause of death and disability worldwide that involves inflammation from its inception to the emergence of complications. Studies have demonstrated that intervention with helminth infections or derived products could modulate the host immune response and effectively prevent or mitigate the onset and progression of inflammation-related diseases. Therefore, to understand the molecular mechanisms underlying the development of atherosclerosis, we intervened in ApoE Atherosclerosis (AS) is a type of low-grade chronic inflammatory disease characterized by abnormal lipid metabolism and the infiltration of inflammatory cells in arterial walls; this is a major global cause of myocardial infarction and stroke . MechaniEpidemiological investigations identified a negative relationship between helminth infection and the incidence of inflammatory diseases and were defined as the “hygiene hypothesis” by Strachan as early as 1989 ,11. ResuAncylostoma duodenale, Necator americanus, and Ancylostoma ceylanicum, can cause chronic blood loss from their digestive tract mucosa and severe anemia in cases of heavy infection [Nippostrongylus brasiliensis (N. brasiliensis), also known as rat hookworm, can infect rodent animals and exhibits a similar life cycle and infection route as human hookworm, thus making it an ideal model for hookworm research [N. brasiliensis infection could induce type two immune responses, manifesting as a high level of anti-inflammatory cytokines, including IL-4, IL-10, and IL-13, which could play a role in tissue repair and the removal of worms in a rodent model [N. brasiliensis, larvae 3 stage (L3)- and larvae 5 stage worm (L5)-derived proteins, could induce an alternative activated macrophage phenotype with anti-inflammation properties in vitro [N. brasiliensis) or its derived products might generate positive effects on AS through induced host anti-inflammation responses.Hookworm, a type of soil-transmitted helminth, is mainly endemic in developing countries within tropical and subtropical regions worldwide . Estimatnfection . A previnfection . Furthernfection . Furthernfection . Collectresearch . Previount model . Our prein vitro . Based o-/- mouse model maintained on a high-fat diet; one experiment targeted AS initiation, while the other targeted the processes involved in progression. Following intervention with N. brasiliensis infection and immunization with its derived products, we analyzed several factors to evaluate interventional effects, including body weight, histopathological alterations in the liver and aortic arch, along with serum levels of lipids and cytokines.To test this hypothesis, two independent animal experiments were designed by using the ApoEN. brasiliensis was gifted by Professor Alex Loukas at James Cook University and has been bred and maintained in our laboratory for approximately five years. Sprague-Dawley rats  were purchased from Gempharmatech Co., Ltd.  and raised by the Animal Center of Jiangsu Institute of Parasitic Diseases  and used as the definitive host for parasite breeding in accordance with a previous protocol [N. brasiliensis was maintained by infecting rats with L3 stage larvae by subcutaneous injection and collecting feces daily from day 6 to day 9. The feces were mixed with appropriate amounts of 5 μg/mL amphotericin solution, carbon powder, and corn cob bedding to form a paste, transferred to absorbent paper in the center of a Petri dish, and incubated at 26 °C. After 1 w, L3 larvae were observed to migrate to the edge of the Petri dish, collected, and washed in PBS.protocol . BrieflyN. brasiliensis were used for the evaluation of efficacy. L3-stage larvae were collected from the Petri dishes of cultured and infected rat fecal samples, as described previously [N. brasiliensis were collected from the small intestine of infected rats as described previously [g for 10 min at 4 °C, the supernatant was collected, quantified with a BCA Protein Assay Kit , and stored at −80 °C to await further analysis.Proteins derived from the L3 and L5 stage larvae of eviously . L5 stageviously . Protein-/-) mouse was used to establish a mouse model of AS; this was induced by feeding with a high-fat diet (1.25% cholesterol) which as purchased from Research Diets . For the AS initiation intervention experiment (-/- mice (5 weeks old) were provided by the Animal Center of JIPD; these were divided randomly into five groups with 8 mice in each group: model group (model), N. brasiliensis infection group (infection), vehicle control group (vehicle), L3 protein group (L3-protein) and L5 protein group (L5-protein). All mice were fed a high-fat diet for 12 weeks. Five hundred L3 larvae were injected subcutaneously into each mouse from the infection group every two weeks. In addition, extracted L3 and L5 proteins were emulsified with an equal volume of Freund’s incomplete adjuvant  and injected subcutaneously every two weeks  into mice in the L3-protein and L5-protein groups, respectively. The same amount of Freund’s incomplete adjuvant was used for each mouse in the vehicle control group with the same time interval. All mice were euthanized at the end of week 12 to evaluate the effects of the intervention.The apolipoprotein-E-deficient  were used and randomly divided into five groups (eight mice per group): model, infection, vehicle, L3-protein, and L5-protein groups. All mice were fed a high-fat diet for 20 weeks. Beginning at 12 weeks, each mouse from the five groups was treated with the same amounts, same injection routes, and intervals  as described for the AS initiation experiment. All mice were euthanized at the end of week 20 to evaluate the effects of the intervention.For the AS progression intervention experiment A, anothehttps://www.olympus.com.cn/) (accessed on 12 July 2022). The photographed images were analyzed, and relative AS plaque area and lipid contents were calculated through ImageJ software (https://imagej.nih.gov/ij/) (accessed on 15 April 2022). For the lipid content of the liver, 3–5 microscopic fields per mouse were selected. A percentage of red lipid droplets over the entire field was quantified and used for statistical analysis.Tissues, including the aortic arch and liver, from all mice from the two experiments were harvested carefully after euthanasia and used for histopathological analysis, which covered plaque areas of AS and lipid contents in the plaque and liver. For aortic arch tissues, we cut 5 μm sections that spanned the entire aortic arch . For liver tissues, the same part of liver tissue was collected from each mouse, and 5 μm slices were cut into 10 sections. Oil Red O staining was carried out for each slide with the Oil Red O Staining Kit  in accordance with the manufacturer’s instructions. The stained slides were observed by microscopy with low-power and high-power fields  and photographed by Cellsens Dimension software  with Cytometric Bead Array (CBA) Flex Sets Kit  according to the kit protocols.Following euthanasia at the end of the two experiments, blood samples were obtained from the eyeballs of each mouse, and serum samples were isolated by centrifugation at 3000× In the AS progression intervention experiment, blood samples were obtained from the eyeballs of each mouse at the end of week 20; the animals had fasted for one day previously. Serum was isolated, as described above, for the detection of lipid levels, including total cholesterol, triglyceride, and low- and high-density lipoproteins, by commercial kits  according to the kit instructions.t-test or one-way ANOVA followed by Dunnett’s multiple comparisons. p < 0.05 was considered statistically significant.GraphPad Software (Version 6.01) was applied for data processing and statistical analysis. Data comparison involved the Student’s N. brasiliensis infection group showed a slower body weight gain. A significant reduction in body weight in the N. brasiliensis infection group was observed when compared to that in the model group in week 12 . Furthermore, a significant decrease in lipid content was also observed in the L3-protein or L5-protein group when compared to that in the vehicle group . The levels of IL-2, IFN-γ, and TNF-α were also detected but showed no significant differences between the two groups (data not shown). When compared to the vehicle group, TNF-α levels in the L3-protein and L5-protein groups were significantly decreased (p < 0.05 and 0.01). IL-6 levels were only significantly increased in the L5-protein group when compared to those in the vehicle group (p < 0.05). Furthermore, there was a significant increase in the levels of IL-4 and IL-10 in the L3-protein and L5-protein groups when compared to those in the vehicle group  intervention during AS initiation in the ApoE-/- mouse model when fed with a high-fat diet.Serum samples were collected at the end of week 12, and the cytokine levels of each mouse were detected by flow cytometry. As shown in N. brasiliensis infection group when compared to that in the model group (p < 0.01). For N. brasiliensis-derived products intervention, there were also significant reductions in liver lipid content and relative plaque areas in the L3-protein and L5-protein groups when compared to that in the vehicle group (p < 0.01). However, there were no significant differences between the L3-protein and L5-protein groups with regard to liver lipid content and relative plaque areas. These results indicated a protective effect manifesting as decreased liver lipid content and contractible plaque area, was induced by N. brasiliensis infection and derived products intervention during the process of AS progression in the ApoE-/- mouse model when fed a high-fat diet.Oil Red O staining of pathological sections was used to evaluate lipid contents in the liver and plaque areas of the aortic arch. As shown in N. brasiliensis infection intervention when compared to the model group . However, a significant elevation of high-density lipoprotein levels was observed following N. brasiliensis infection intervention when compared to the model group (p < 0.001). For N. brasiliensis-derived products interventions, there were no significant differences in the serum levels of total cholesterol and triglyceride in the L3-protein and L5-protein groups when compared to the vehicle group (p < 0.0001 and <0.001). No significant difference was observed between the L3-protein and L5-protein groups. Furthermore, there was a significant elevation of the serum levels of high-density lipoprotein in the L3-protein group (p < 0.05); however, there was no significant difference when compared between the L5-protein group and the vehicle group  and increase protective high-density lipoprotein levels during the process of AS progression in the ApoE-/- mouse model fed a high-fat diet.Serum samples were collected from each mouse at the end of week 20, and fasting serum lipid levels were analyzed. As shown in le group F,G. Howele group I. ResultN. brasiliensis infection intervention, there were significant decreases in the levels of IFN-γ and IL-2 in the N. brasiliensis infection group when compared to those in the model group (p < 0.01). There were significant elevations of TNF-α, IL-6, and IL-10 levels in the N. brasiliensis infection group when compared to those in the model group . No significant difference in IL-4 levels was observed between the two groups (data not shown). For N. brasiliensis-derived products intervention, there were significant decreases of IL-2 and IL-6 levels in the L3-protein and L5-protein groups when compared to the vehicle group . Furthermore, there was a significant elevation of IL-10 levels in the L3-protein and L5-protein groups when compared to the vehicle group (p < 0.001 and <0.001). No significant differences between IFN-γ (data not shown) and TNF-α (N. brasiliensis infection and derived products interventions could decrease pro-inflammation cytokine levels (IL-2 and IFN-γ) and increase anti-inflammation cytokine levels (IL-10) during the process of AS progression in the ApoE-/- mouse model fed a high-fat diet.Serum samples were collected from each mouse at the end of week 20, and serum levels of IL-2, IFN-γ, TNF-α, IL-6, IL-4, and IL-10 were detected by flow cytometry as described above. For nd TNF-α K were obN. brasiliensis infection and derived product intervention (immunization) for AS in the ApoE-/- mouse model for the first time. Results showed that N. brasiliensis infection and derived products intervention could prevent and alleviate the development of AS, manifesting as lower weight increases, smaller areas of AS plaques, lower levels of lipid content in the liver and aortic arch and higher levels of anti-inflammation cytokines in the serum.It has been widely accepted that helminth infection or helminth-derived products could be beneficial for inflammatory diseases and might provide a novel approach to the prevention or treatment of disease ,27,28,29Trichuris suis), human hookworm (Necator americanus), and rat tapeworm (Hymenolepis diminuta), thus demonstrating a therapeutic role in autoimmune, inflammatory, and allergic diseases [N. brasiliensis infection as a model of human hookworm to intervene in AS and observed a protective role for AS initiation and progression. Due to self-cure from N. brasiliensis infection within two weeks in the mouse model, we carried out multiple infections to sustain the infection conditions in our two animal experiments. Nonetheless, we still observed a weakened physical condition in the infected mice (data not shown), which may have influenced the final identification of protective effects. For safety and ethical consideration, screening the helminth-derived molecules with biological activities would be more suitable for worm therapy in the future; a variety of helminth-derived molecules have been screened and shown to exert different biological activities for disease intervention [N. brasiliensis-derived products, L3- and L5-derived proteins, through a typical immunization procedure; these had a similar protective effect to N. brasiliensis infection, thus providing possibilities for screening and evaluation helminth-derived biologically active molecules for AS intervention in the future. However, translation from rodent hookworm to human hookworm is another key consideration in subsequent research. Furthermore, N. brasiliensis-derived excretory-secretory products could be another important source for active molecule screening; these contain various anti-inflammatory metabolites, as demonstrated previously; these could be investigated for AS intervention in the near future [Direct helminth infection, also known as worm therapy, was first proposed in 1968, thus demonstrating their importance for human health . Numeroudiseases ,34,35. Irvention ,36. In tr future ,37.N. brasiliensis-derived products (L3 and L5 proteins) could induce M2 macrophage polarization in vitro [Mechanisms of AS initiation and progression have not been fully elucidated until now, although low-grade chronic inflammation was thought to play an important role . Anti-inin vitro ; whetherin vitro ,42; thisN. brasiliensis infection and derived products intervention. Furthermore, decreased levels of serum lipoprotein were observed, including total cholesterol, triglyceride, and low-density lipoprotein, following intervention; this indicated that N. brasiliensis and derived products could reduce serum lipoprotein. In contrast to LDL, high-density lipoprotein (HDL) is considered to be beneficial for blood cholesterol and can protect against AS [N. brasiliensis and derived products intervention; this could further induce the protective effects against AS development. However, the mechanisms associated with the alterations of serum lipoprotein levels and different interventions need to be further investigated.High serum lipoprotein levels, especially elevated low-density lipoprotein (LDL) levels, are known to be strongly associated with AS development ,44. AS lainst AS . Our resN. brasiliensis and derived product intervention in a mouse model, possibly via anti-inflammatory and lipid-lowering approaches, thus providing new insight for the prevention and treatment of atherosclerosis.In summary, a protective effect was confirmed for atherosclerosis initiation and progression by


The aim of this study was to determine whether increases in post-exercise endocrine response to low-load resistance exercise with blood flow restriction and high-load resistance exercise would have association with increases in muscle size and strength after an 8-week training period. Twenty-nine untrained men were randomly allocated into three groups: low-load resistance exercise with (LL-BFR) or without blood flow restriction (LL), and high-load resistance exercise (HL). Participants from LL-BFR and LL groups performed leg extension exercise at 20% of one repetition maximum (1RM), four sets of 15 repetitions and the HL group performed four sets of eight repetitions at 80% 1RM. Before the first training session, growth hormone (GH), insulin-like growth factor 1 (IGF-1), testosterone, cortisol, and lactate concentration were measured at rest and 15 min after the exercise. Quadriceps CSA and 1RM knee extension were assessed at baseline and after an 8-week training period. GH increased 15 min after exercise in the LL-BFR (p = 0.032) and HL (p < 0.001) groups, with GH concentration in the HL group being higher than in the LL group (p = 0.010). There was a time effect for a decrease in testosterone (p = 0.042) and an increase in cortisol (p = 0.005), while IGF-1 remained unchanged (p = 0.346). Both muscle size and strength were increased after training in LL-BFR and HL groups, however, these changes were not associated with the acute post-exercise hormone levels (p > 0.05). Our data suggest that other mechanisms than the acute post-exercise increase in systemic hormones induced by LL-BFR and HL produce changes in muscle size and strength. Low-load resistance exercise (20 to 30%) of one repetition maximum (1RM) in combination with blood flow restriction (LL-BFR) has been demonstrated to induce increases in muscle size and strength to a similar level to that observed with high-load resistance exercise (≥65-70% 1RM) .Localized hypoxia seems to be the primary candidate mechanism responsible by the anabolic response to LL-BFR . In thisImportantly, although the alleged anabolic role of acute endocrine responses to conventional resistance training has been strongly challenged , little Twenty-nine physically active male college students were randomly allocated into three groups: low-load resistance exercise with blood flow restriction , low-load resistance exercise , and high-load resistance exercise . Participants were not engaged in any form of regular physical training and had no previous experience in strength training. They were free from any musculoskeletal disorders and drug or nutritional supplements ingestion. Participants were informed of the benefits and potential risks of the investigation and gave their written informed consent prior to the commencement of the study. The study was conducted according to the Declaration of Helsinki and the University's Research Ethics Committee approved the experimental protocol.After two familiarization sessions with a bilateral knee extension exercise, a one repetition maximal test (1RM) and blood flow restriction protocols, participants from LL, LL-BFR, and HL groups performed their respective training protocols. Blood samples were taken before the first session of exercise (rest) and 15 min after completing the exercise session (15 min) for determination of GH, IGF-1, testosterone, cortisol, and lactate concentrations. The quadriceps cross-sectional area (CSA) and knee extension 1RM were assessed before and after 8 weeks of training by magnetic resonance imaging technique. Pearson´s correlation coefficients were calculated to determine the relationship between the acute absolute changes of hormones and lactate concentrations and CSA and 1RM measurements.Prior to testing, participants rested in a supine position for at least 30 min, and then, blood samples were collected at 8:00 - 10:00 a.m. using an indwelling heparin lock catheter which was inserted into the superficial antecubital vein of the left arm for the determination of GH, IGF-1, testosterone, and cortisol concentrations. Blood samples were taken before starting the exercise (rest) and 15 min after the exercise session (15 min). For hormone measurements, blood samples (5 ml) were drawn into test tubes containing EDTA. All blood samples were kept in ice-cold water and centrifuged (10000 rpm) for 10 min and the plasma was removed and stored frozen at - 80°C until the assays were performed. Plasma hormone concentrations were determined by a commercially available enzyme-linked immunosorbent assay (ELISA). Data of GH of five participants from three groups were excluded from the analysis due to technical error: two from the LL group, one from the LL-BFR group and two from the HL group. In addition, two participants of the HL group were excluded from IGF-1 analysis.Measurements of blood lactate concentration were analysed for each experimental protocol at rest and 15 min after exercise. After local asepsis with alcohol, a small earlobe incision was done using a disposable lancet to collect the blood. An arterial blood sample (25μl) was collected and inserted in a capillary tube. Blood lactate concentrations were measured by an electro-enzymatic lactate analyzer , which was previously calibrated with a known concentration of lactate of 5 mmol∙L-1.The quadriceps CSA was obtained through magnetic resonance imaging  and measured at 50% of the femur length. The images were then transferred to a workstation  and were traced by a specialized researcher. Mean values from three measures were used for further analysis.-1, one set of eight repetitions at approximately 50% 1RM was performed, followed by the second set of three repetitions at approximately 70% 1RM with a 2 min rest interval between trials. After that, participants were allowed up to five attempts to achieve their 1RM with a 3-min rest interval between trials. The greatest 1RM weight lifted was considered for further analysis.For knee extension 1RM determination, after participants ran for 5 min on a treadmill at 9 km∙hParticipants were asked to lie in a supine position while resting comfortably. A vascular handheld Doppler probe was placed over the tibial artery to capture its auscultatory pulse. For the determination of blood pressure (mmHg) necessary for a complete vascular restriction (pulse elimination pressure), a standard blood pressure cuff was attached to the participant’s thigh  and then inflated up to the point in which the auscultatory pulse was interrupted . The cuf-1 for five minutes, participants from LL, LL-BFR, and HL groups performed bilateral knee extension protocols. Participants from LL and LL-BFR groups performed four sets of 15 repetitions at 20% 1RM. For the LL-BFR protocol a nylon cuff (17.5 cm width x 92 cm length) was placed at the most proximal region of the thigh and inflated at 80% of arterial occlusion pressure and removed immediately after the protocol. Participants from the HL group performed four sets of eight to ten repetitions at 80% 1RM. The LL and LL-BFR protocols were performed with a 60 s rest interval between sets, while the HL protocol was performed with a 90 s rest interval between sets. All protocols were performed with a tempo of 1 s for the concentric and 1 s for the eccentric phases. Participants performed training sessions twice a week, for 8 weeks, with supervision of an experienced researcher. Only one participant withdrew from the HL group for personal reasons.After a general warm-up on a treadmill at 9 km∙hp ≤ 0.05, and all analyses were conducted with the statistical software package SAS 9.2. .Data are presented according to descriptive statistics as means and standard deviations. The Shapiro-Wilk test was used to assure that the data was normally distributed. A mixed model for repeated measures was applied, using as fixed factors the experimental groups  and times (pre- and 15 min post-exercise) for acute hormonal responses  and lactate concentrations. When a significant F value was obtained, the Tukey post hoc test was used for multiple comparisons. Pearson´s correlation coefficients were calculated to determine the relationship between the acute absolute changes (pre- and 15 min post-exercise) in the measured variables  with chronic absolute changes (pre- and post-8-week training) in the CSA (ΔCSA) and 1RM (Δ1RM). The level of significance was set at p = 0.037). GH concentration was increased 15 min after exercise in the LL-BFR (p = 0.032) and HL (p = 0.0009) groups (p = 0.010). There were no significant condition x time interactions for testosterone and cortisol, but there was a main effect of time for testosterone (p = 0.042) and cortisol (p = 0.005) (There was a significant time x condition interaction for GH  groups . In addi= 0.005) . There w= 0.005) .p = 0.016). Lactate concentrations were increased 15 min after exercise in the LL, LL-BFR, and HL groups (p = 0.0001); however, lactate concentration was significantly higher in the LL-BFR than in the LL group 15 min after exercise (p = 0.008) (There was a significant time x condition interaction for lactate concentration (= 0.008) .p = 0.965; LL-BFR pre: 7720 ± 1298, post: 8182 ± 1230, p < .0001; HL pre: 7572 ± 1832, post: 8030 ± 1983 mm2, p < .0001) and 1RM . Moreover, increases in muscle mass (~6.3% vs. 6.1%) and strength (40% vs. 36%) were similar in the LL-BFR and HL groups, respectively, whereas LL resulted in much smaller strength increments (~23%) with no changes in the CSA. However, there was no significant difference between groups in the muscle CSA and 1RM (p > 0.05).As expected, 8 weeks of training resulted in significant gains in the muscle CSA . Lactate concentration was significantly correlated with Δ1RM and ΔCSA after the training period in the LL group . Finally, we observed a similar response pattern between LL-BFR, LL, and HL when considering IGF-1, testosterone, and cortisol concentrations, with no association between training-induced changes in systemic hormones, muscle size and strength adaptations in none of the groups.For years, the contention was that acute post-exercise  responses of hormones such as GH, IGF-1, and testosterone were important drivers of muscle growth. However, even though this assertion is supported by some , other sIn the present study, there was an increase in lactate concentration in all groups and an increase in GH concentration 15 min after exercise in the LL-BFR and HL groups. In fact, these findings corroborate some other studies , howeverTestosterone decreased 15 min after exercise in all groups. In addition, there was no association between post-exercise changes in testosterone and the CSA or 1RM, demonstrating that despite a significant reduction in testosterone concentration, muscle size and strength gains were still unaffected, supporting our hypothesis that training-induced gains in muscle mass and strength are not determined by post-exercise hormonal response. Furthermore, similar to our findings, previous studies have investigated the testosterone response to LL-BFR and found no significant changes . In thisThere was a positive relationship between acute exercise-induced changes in cortisol and training-induced changes in muscle size in the HL group. This effect has been previously observed and may be related to a stress response associated with high-load resistance exercise . For exaThe current data are consistent with the notion that local mechanisms within skeletal muscle are the primary drivers of muscle growth resulting from resistance exercise and are dissociated from post exercise-induced hormonal responses. For example, a decrease in mRNA gene expression of myostatin and an increase in mRNA gene expression of follistatin isoforms, GASP-1 and SMAD-7 via mechanotransduction pathways may play a significant role .A limitation of this study is that the hormonal concentrations were assessed only at one time point (pre-training). It is possible that these hormones may have changed across time, however, previous studies have demonstrated no change in the time course of systemic hormones  and thesIn conclusion, the present study suggests that despite transient exercise-induced increases in systemic endogenous hormones, these changes did not correlate to the enhanced muscle size and strength observed after LL-BFR. Additionally, hormonal, phenotypical , and functional  responses to LL-BFR were comparable to those observed after HL. Finally, all these changes occurred in the absence of significant post-exercise increases in either IGF-1 or testosterone concentrations. These findings reinforce the lack of evidence that post-exercise-induced hormonal changes are important in regulating skeletal muscle hypertrophy and strength gains following blood-flow-restriction and high-load resistance training.


Corynebacterium glutamicum is regarded as an industrially important microbial cell factory and is widely used to produce various value-added chemicals. Because of the importance of C. glutamicum applications, current research is increasingly focusing on developing C. glutamicum synthetic biology platforms. Because of its ability to condense with adipic acid to synthesize the industrial plastic nylon-46, putrescine is an important platform compound of industrial interest. Developing a high-throughput putrescine biosensor can aid in accelerating the design–build–test cycle of cell factories (production strains) to achieve high putrescine-generating strain production in C. glutamicum. This study developed a putrescine-specific biosensor (pSenPuuR) in C. glutamicum using Escherichia coli-derived transcriptional factor PuuR. The response characteristics of the biosensor to putrescine were further improved by optimizing the genetic components of pSenPuuR, such as the response promoter, reporter protein, and promoter for controlling PuuR expression. According to the findings of the study, pSenPuuR has the potential to be used to assess putrescine production in C. glutamicum and is suitable for high-throughput genetic variant screening. Putresc®, DSM) . Putreschia coli  and Coryutamicum ,8,9,10.  pathway ,11.Traditional microbial-metabolic-engineering methods require extensive optimization and the reconstruction of the metabolic network to create putrescine-producing strains with high yielding ability. The entire process is time-consuming and labor-intensive ,12,13,14C. glutamicum. [puu genes in E. coli K12 that metabolize putrescine to gamma-aminobutyric acid via gamma-glutamylation intermediates [puuA and puuD genes. PuuR binds to the gene spacer between puuA and puuD and inhibits puu gene expression at low intracellular putrescine concentrations. When putrescine concentration is high, PuuR dissociates from DNA and the puu gene is transcribed, thereby initiating the putrescine utilization pathway. Chen et al. recently developed a PuuR putrescine-responsive biosensor in E. coil [Metabolite response TFs are primarily involved in metabolite transport and utilization pathways. PsenPut is a diamine biosensor based on the TF CgmR (CGL2612), a TetR-family regulatory protein that regulates the endogenous putrescine transport pathway in tamicum. . The devmediates . PuuR ha E. coil . When thE. coli-derived transcriptional factor PuuR as a high-throughput genetic tool for C. glutamicum in this study. The E. coli-derived TF PuuR has demonstrated the highly specific detection of putrescine among other diamines and amino acids in the diamine synthesis pathway. We further optimized the genetic components of the putrescine biosensor pSenPuuR to improve the signal-to-noise ratio and sensing sensitivity of the biosensor. Finally, we validated the utility of the optimized putrescine biosensor by using different ODCs obtained from the initial phylogenetic tree. The findings indicate that pSenPuuR could be used to monitor putrescine biosynthesis in C. glutamicum and could be useful for ODC HTS.We aimed to create a putrescine-specific biosensor (pSenPuuR) based on the E. coli TOP10 was grown in a Luria–Bertani medium  at 200 rpm and 37 °C for plasmid construction. C. glutamicum ATCC 14067 (bought from ATCC) was grown in a BHI liquid medium ) at 220 rpm and 30 °C. The fermentation medium contained 80 g of glucose, 7.5 g of urea, 10 g of soya peptone, 0.5 g of MgSO4, 0.7 g of KH2PO4, and 42 g of MOPS (per liter). For shake flask fermentation of putrescine-producing strains, transformants were selected, picked from the plates, and inoculated individually in 50 mL Erlenmeyer flasks containing 5 mL of the BHI liquid medium, then cultured for 12 h at 30 °C with 220 rpm shaking. The starting optical density (OD) was diluted to 0.2, and the cultures were centrifuged for 2 min at 6000× g rpm. The BHI liquid medium was then aspirated, and the cells were resuspended with fermentation medium without glucose and urea. Each culture was then inoculated into 500 mL Erlenmeyer flasks with 50 mL of fermentation medium. The following antibiotics were added at the following concentrations where appropriate: kanamycin at 50 mg/L or tetracycline at 10 mg/L for E. coli and kanamycin at 25 mg/L or tetracycline at 5 mg/L for C. glutamicum. In addition, 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) was used to induce ODC expression. puuAS, PpuuAF, PpuuDS, and PpuuDF were amplified from the E. coli genome. PuuR was cloned into PEC-XK99E to obtain pEC-XK99E-PuuR, and the response promoters with the reporter protein mCherry (GenBank: MZ027319.1) were cloned into PEC-T18-mob2. This finally resulted in T18-PpuuAS-mCherry, T18-PpuuAF-mCherry, T18-PpuuDS-mCherry, and T18-PpuuDF-mCherry, respectively. To construct a single-plasmid sensor system, the PuuR linear expression cassette Ptrc-PuR-rrnBT1T2 with Ptrc as promoter and rrnBT1T2 as terminator was amplified from pEC-XK99E-PuuR and assembled by homologous recombination into the T18-PpuuDF-mCherry plasmid in the opposite direction of reporter protein transcription. Following that, we created a series of plasmids to optimize the genetic components of the sensor. The backbone was the single-plasmid sensor pSenPuuR. The original mCherry gene downstream of PpuuDF was replaced with egfp (GenBank: AFA52654.1) and sfgfp (GenBank: UFQ89826.1), yielding the plasmids pSenPuuREGFP and pSenPuuRsfGFP. To control PuuR expression, the pSenPuuRsfGFP plasmid was used as the backbone, and the original promoter Ptrc was replaced with various native promoters of C. glutamicum. The plasmids that obtained were designated as pSenPuuRsfGFP-Psod, pSenPuuRsfGFP-PcspB, pSenPuuRsfGFP-Pddh, and pSenPuuRsfGFP-PaspB. The pEC-XK99E plasmid was used to overexpress ODCs involved in putrescine production. PCR products of all linear vectors, reporter proteins or promoter fragments were first obtained by KOD DNA polymerase , and then assembled using NEB Builder HiFi DNA Assembly Master Mix . At first, a two-plasmid sensor system was constructed. The TF PuuR (GenBank: NP_415815.1) and its corresponding response promoters PA single colony of the recombinant strain containing the sensor’s associated plasmid was picked from the plate, inoculated into 5 mL of BHI medium (containing 5 mg/L tetracycline and/or 25 mg/L kanamycin as appropriate) and cultured at 30 °C for 12 h while shaking at 220 rpm. The cells were then diluted 1:100 into a 48-well plate containing 900 µL of fresh BHI medium and cultured for 24 h in a microplate thermostatic oscillator at 30 °C while shaking at 800 rpm. To induce PuuR expression for fluorescence detection of recombinant strains with two-plasmid sensors, 0.1 mM IPTG was added. To quantify the dose response of the sensor to exogenous putrescine, the seed culture of Cg14067/pSenPuuR was inoculated into 900 ul of fresh BHI and added with tetracycline and different concentrations of putrescine (0–100 mM). The corresponding antibiotics and 20 mM ligand compound were added according to the experimental needs to evaluate the sensor ligand’s specificity. After the culture was completed, 200 µL of culture medium was taken from each well, and the cells were washed twice with phosphate-buffered saline  and resuspended in the same buffer. The fluorescence intensity of the red fluorescent protein mCherry, the green fluorescent protein GFP, and the cell density were measured in the 96-well plates using a BioTek microplate reader and 200 µL of the resuspension solution was used. The wavelengths of excitation and emission for red fluorescent protein and green fluorescent protein were 584/610 nm and 488/520 nm, respectively.C. glutamicum ATCC 14067 (Cg14067). Next, the self-excisable linear expression cassettes dsDNA-Peftu-PargC and dsDNA-ΔargF were constructed according to the RecET-Cre/loxP system [argCJBD promoter on the genome  was transformed into strains P and PΔ to obtain PUTP and PUTPΔ, respectively. TTTG was used as the PAM sequence in the CRISPR/Cpf1&RecT-based genome fine modification method [snaA gene was replaced with TAA to terminate SnaA expression at an early stage. To complete the point mutation of the snaA gene, the PΔ/pJYS1Ptac receptor strain was transformed with the crRNA expression plasmid pJYS2_snaA and the 59 bp lagging oligonucleotide O-snaA. To obtain strain PΔD, recombinant C. glutamicum containing double plasmids was incubated overnight in an antibiotic-free BHI liquid medium at 34 °C with 220 rpm shaking. Finally, the recombinant strain PUTPΔD was created by transferring pEC-XK99E-SpeCEC into PΔD  were added to the crush tube before the cells were resuspended in 1.5 mL of PBS solution and lysed with Bead Ruptor 12 . The samples were centrifuged at 14,000× g rpm for 3 min at 4 °C after 10 cycles of lysis. Finally, 1 mL of the cell-free lysate in the supernatant was slowly aspirated for putrescine quantification. The detection of putrescine was performed by precolumn derivatization with danthanoyl chloride, and the derivatized product was analyzed using high-performance liquid chromatography  equipped with an ultraviolet detector  at 254 nm using a Sunfire C18 . Acetonitrile and water  were used as the mobile phase, which was maintained at a flow rate of 1 mL/min and a temperature of 35 °C [After the fermentation of the recombinant strain, 500 µL of the fermentation broth was taken from each sample and centrifuged at 12,000× of 35 °C .Enterobacter cloacae (SpeFECL) had the highest specific ODC activity in C. glutamicum [ECL was chosen as the probe sequence to mine more ODCs from the National Center for Biotechnology Information (NCBI) database. BlastP results with a candidate ODC sequence revealed similarities ranging from 50% to 90%. One ODC with a similarity of more than 90% was also kept. Some repeated or truncated sequences were removed, leaving 8245 candidate sequences for phylogenetic tree construction. A comparison file spec.fasta was generated by sequence matching of ODCs using MAFFT, and the final spec.tree file was generated by the FastTree construction of a maximum-likelihood phylogenetic tree. The crystal structure of Lactobacillus 30a ODC dimer (PDB:1ord) was used as a template by the modeling module function of SWISS-MODEL to obtain the model structure of the candidate sequences, and the pdb file was downloaded. The candidate protein model was docked onto the ornithine molecule with a radius of 12 using the semi-flexible docking software program CDOCKER in Discovery Studio 3.0. The A-chain active sites N153 (N161) and T387 (T395) were chosen, while the B-chain active sites H216 (H224) and Y652 (Y660) were chosen. To assess the reliability of the ligand-protein docking and binding stability, the total energies of the receptor and ligand -CE (-CDOCKER ENERGY) and the receptor–ligand interaction energy -CIE (-CDOCKER INTERACTION ENERGY) were used. The lower the CE value, the less total energy consumed during the docking process and the more stable the docking system. The lower the CIE value, the better the ligand–receptor interaction binding. ODCs with -CE or -CIE significantly higher than that of the probe enzyme were selected from the candidate sequences based on the energy ranking.Currently, phylogenetic tree analysis has become an important strategy for mining new functional enzymes. In order to enrich the ornithine decarboxylase in the putrescine synthesis pathway, we preliminarily screened the ornithine decarboxylase via phylogenetic tree and molecular docking. This method requires a protein sequence with a known function as a probe. Then, the candidate sequences were screened from the huge sequence information by analyzing the affinity of phylogenetic evolution. Finally, the screening range was further narrowed by molecular docking. Li et al., compared seven ODCs from different sources and found that the ODC from utamicum . TherefopuuA and puuD, and the gene spacer region between puuA and puuD contains four DNA-binding motifs; thus, four response promoters were chosen, namely the full-length promoters of puuA and puuD (PpuuAF and PpuuDF) and the truncated promoters of puuA and puuD , with mCherry being the reporter protein to test the dynamic range of the sensor . Based on this, competent cells were prepared, and the regulator plasmid pEC-XK99E-PuuR was transformed into each of the transfected strains  to obtain Cg14067AF/R, Cg14067AS/R, Cg14067DF/R, and Cg14067DS/R, respectively, simulating the repressed state (“OFF” state) of the response promoters in C. glutamicum. The fluorescence-to-OD ratios of the recombinant strains were measured after 24 h of incubation at 30 °C under shaking at 800 rpm in 48-well plates. The dynamic range of the promoters was defined as the fluorescence-to-OD ratios of the recombinant strains measured in the derepressed state to that measured in the repressed state (ON/OFF). The fluorescence-to-OD ratios of the response promoters PpuuAF, PpuuAS, PpuuDF, and PpuuDS in the derepressed state were all higher than those in the repressed state by PuuR, as shown in puuDF was thus chosen as the response promoter for the next sensor design.PuuR can bind to the promoter regions of e sensor a. First,puuDF-mCherry and the regulator plasmid pEC-XK99E-PuuR, the levels of IPTG and putrescine in the BHI culture medium were independent of each other  in the medium. When there is no putrescine in the culture medium, PuuR binds to the response promoter PpuuDF, preventing mCherry expression. Putrescine binds to PuuR when a certain amount of putrescine is added to the culture medium. Meanwhile, PuuR was released from the promoter PpuuDF, allowing mCherry expression , and the remaining promoters were not significantly different . However, under 100 mM putrescine concentration, the maximum fluorescence levels were very different, with Psod and Pddh having the lowest fluorescence levels and PcspB and Ptrc having the highest levels  of the L-ornithine synthesis pathway of L-arginine was knocked out. The recombinant strain PUTPΔ had a putrescine accumulation of 28.7 mM at the shake flask level after overexpressing ODC from E. coli (Metabolic engineering of Cg14067 for putrescine production was carried out using CRISPR/Cpf1&RecT and RecET-Cre/ E. coli . Therefo E. coli .ECL (Protein No. WP_013097558.1) was used as the probe sequence and was searched using BlastP in the NCBI database. We obtained 8245 candidate ODCs for phylogenetic tree analyses after filtering and screening (ECL branch (ECL branch (The amino acid sequence of SpeFcreening . A totalL branch . Ten ODCL branch , and thiL branch . The perL branch .s of 0.67 (p = 0.0025; We introduced the most sensitive plasmid pSenPuuRsfGFP-Ptrc into the previously constructed strain PΔD to demonstrate the application of the PuuR-based putrescine biosensor system in metabolic engineering. The phylogenetic tree screening ODCs ODC1, ODC2, ODC3, ODC4, ODC5, and ODC6 were then cloned into the IPTG-induced pEC-XK99E and transformed into the recombinant strain PΔD/pSenPuuRsfGFP-Ptrc. After 12 h of fermentation, we collected the cells and measured the fluorescence-to-OD ratio of the strains as well as the intracellular putrescine concentration a,b. The s of 0.63 (p < 0.0001; We also investigated whether the biosensor could be used to report the concentration of putrescine in the fermentation supernatant. We measured the concentration of putrescine in the fermentation supernatant after 12, 24, 48, and 72 h. At each of these four time points, we measured the fluorescence-to-OD ratio of the cells. As shown in Putrescine is a diamine that can be used as a monomer to synthesize nylon-46, an excellent industrial plastic ,36,37. CC. glutamicum based on the E. coli-derived TF PuuR. At a high concentration of ligand, the biosensor responded significantly to C4 putrescine, weakly to C3 1,3-propylenediamine, and not at all to diamines above C4 and intermediate amino acids. We systematically optimized the response promoter, reporter protein, and the expression levels of the TF of pSenPuuR to improve the biosensor’s dynamic range and response characteristics. PpuuDF was the response promoter of the optimal putrescine biosensor pSenPuuRsfGFP, sfGFP was the reporter protein, and Ptrc was the promoter controlling PuuR expression. The results also demonstrate that optimizing the genetic components of the biosensor is critical to improving the response characteristics of the biosensor.We developed and optimized a putrescine-specific biosensor in ECL amino acid sequence as a probe, combined with the phylogenetic tree and molecular docking strategy. Finally, we chose six different ODCs to put the optimized putrescine biosensor pSenPuuRsfGFP-Ptrc to the test in metabolic engineering. We looked at the relationship between fluorescence ratio and intracellular putrescine concentration at 12 h and extracellular putrescine concentration at 12, 24, 48, and 72 h. The results revealed a strong Spearman’s correlation between the fluorescence ratio and putrescine production in both vivo and vitro, with correlation coefficients of 0.67 and 0.62, respectively. The putrescine-specific biosensor built in C. glutamicum can detect putrescine production during shaker fermentation and is appropriate for ODC HTS.We obtained 53 ODC sequences with different homologies using the SpeFC. glutamicum have been established, and while endogenous TFs are well adapted, the variety of metabolites that can be detected is limited [Currently, various endogenous TF-based metabolite biosensors in  limited ,27. Ther limited ,40. TFB  limited ,42. Howe limited .


The results suggest that 24-Hour Movement Behaviours have worsened among young people 1-year after Covid-19 pandemic compared to pre-pandemic period. Moreover, the physical activity benefits associated in terms of academic performance seem to have disappeared because of the Covid-19 pandemic. Therefore, there is a public health problem that requires priority and coordinated action by schools, policy makers, and researchers to mitigate the adverse effects of the pandemic on 24-Hour Movement Behaviours.Most studies have shown a decline in the adherence to 24-Hour Movement Guidelines because of Covid-19 lockdown. However, there is little evidence regarding changes 1-year after the pandemic in these guidelines and their possible impact on academic performance. The study aims were: (1) to examine the possible changes in 24-Hour Movement Guidelines for youth  before and after 1-year into the Covid-19 pandemic, and (2) to examine the possible changes in the relationship between 24-Hour Movement Behaviours  and academic performance before and after 1-year into the Covid-19. This is a repeated cross-sectional study in two different samples of young Spanish at different times. Firstly, a total of 844 students  completed a series of valid and reliable questionnaires about physical activity levels, recreational screen time, sleep duration and academic performance before Covid-19 pandemic (March to June 2018). Secondly, a different sample of 501 students  completed the same questionnaires 1-year after Covid-19 pandemic (February to March 2021). Adherence to the three 24-Hour Movement Guidelines was significantly lower 1-year after into the Covid-19 pandemic (0.2%) than before the pandemic (3.3%), while adherence to none of these three recommendations was significantly higher 1-year after the Covid-19 pandemic (66.3%) than before the pandemic (28.9%). The positive relationship between physical activity levels and academic performance was no longer significant after 1-year into Covid-19 pandemic (β = − 0.26;  In early March, Spain experienced an initial increase of 4,200 Covid-19 cases, reaching a peak of 9,181 cases on 26th March 20212. To stop its spread, on 14th March 2020, the Spanish government (and other nations worldwide) declared a state of alarm followed by lockdown measures (from 14th March to 4th May 2020), limiting people's mobility , and forcing the closure of many institutions such as schools, high schools, and kindergartens. This also resulted in the cessation of organised sports and recreational activities and limited access to outdoor places such as playgrounds and parks4.On 11th March 2020, the World Health Organization5. A decrease in Covid-19 cases in Spain in the late spring and summer months led to a reduction in containment measures. In September 2020, most schools and high schools in Spain reopened their doors, using different safety measures such as 1.5 m distance, not sharing materials or the use of face masks. Sports and recreational activities were also resumed with new safety protocols6. Therefore, while all these public health restrictions were necessary to reduce the spread of Covid-19, the health measures adopted by Spain and other countries globally has been negatively related to physical, psychosocial, and cognitive health8.Restrictions or closure measures of Covid-19 meant prohibiting children and adolescents from leaving home for 6 or more weeks at a time, as well as replacing face-to-face lessons with home schooling and online learning activities8. Particularly, there is clear evidence that high physical activity levels9, low recreational screen time10, and optimal sleep duration11, contribute to the overall health and youth development. Meeting these three 24-Hour Movement Guidelines for children and adolescents  could even maximize those health benefits in young people12. However, the systematic review and meta-analysis conducted by Tapia-Serrano et al.13 reported that only 2.68% of adolescents met all three 24-Hour Movement Guidelines, while 28.59% did not meet any recommendation. The limited number of studies carried out in Spain indicated that between 1.7% and 5.4% of adolescents met all three recommendations, while between 8.7% and 10.2% did not comply with any recommendation15.Several authors have suggested that promoting a healthy lifestyle could prevent the adverse health effects of Covid-1916 showed a decline of physical activity levels, an increase in recreational screen time, and irregular sleep patterns, particularly among adolescents, because of Covid-19-related restrictions. Particularly, in Spain, there were only three studies that compared the 24-Hour Movement Guidelines in children and adolescents before and during Covid-19 closure19. The results of these investigations showed negative effects of Covid-19 closure on meeting physical activity recommendations (34.6% to 60.0% before closure vs. 26.5% vs. 51.0% during closure) and sedentary behaviours, especially screen recreational behaviours guidelines (2.5% to 5.0% before closure vs. 1.8% vs. 2.4% during closure), but not on sleep duration guidelines (57.3% to 84.4% before closure 66.6% vs. 84.8% during closure). Nevertheless, most of the studies included in this scoping review examined changes in 24-Hour Movement Guidelines before and during Covid-19 lockdown16.The scoping review conducted by Paterson et al.20. In this study, there were significantly fewer adolescents meeting the three 24-Hour Movement Guidelines during the autumn 2020 than before Covid-19 (5.5% vs. 1.1%). In addition, a significantly higher number of adolescents did not comply with any of the three recommendations during the autumn 2020 than before Covid-19 (50% vs. 17.1%). Specifically, a decrease in physical activity and sleep duration recommendations was identified, while there was no change in screen time guidelines20. However, these findings are not extensible to the rest of the world, as they only looked at children and adolescents in Montreal (Canada). Therefore, further research is required to examine the long-term impact of Covid-19 on 24-Hour Movement Guidelines of youth16.To our knowledge, there is only one study that have examined trends of 24-Hour Movement Guidelines before and after approximately 1-year into the Covid-19 pandemic21. Given the adoption of a healthy lifestyle has been positively associated with brain development processes, cognitive function, and academic performance23, one would expect that the decline in 24-Hour Movement Guidelines would have negatively affected academic performance. Particularly, previous researches have shown that higher physical levels24, lower recreational screen time25, and optimal sleep duration11 have been positively and independently related to academic performance. To the best of our knowledge, no previous studies have examined the possible changes in the relationship between these 24-Hour Movement Behaviours  and academic performance before and 1-year after into the Covid-19 pandemic and, therefore, further researches are also required. It is important to know whether this relationship may have been altered by the Covid-19 pandemic, as, for example, the type of physical activity may be different  or the quality of sleep may have been impaired. Therefore, perhaps the relationship between 24-Hour Movement Behaviours and academic performance could have been altered because of Covid-19 pandemic.Moreover, a previous systematic review showed a negative effect of school closures on academic performance26 that have shown a decline in adherence with the 24-Hour Movement Guidelines before and during Covid-19 closure, is also expected to be lower 1-year after the Covid-19 pandemic. The second objective was to examine whether the relationship between 24-Hour Movement Behaviours and academic performance was different before and 1-year after the Covid-19 pandemic. Because a decline in adherence to the 24-Hour Movement Guidelines has been observed21, the first hypothesis of the study was that adherence to these recommendations has declined 1-year after the Covid-19 pandemic. With regard to the second hypothesis, because Covid-19 may have altered 24-Hour Movement Guidelines16, it is expected that the relationship with academic performance may have changed after 1-year into the Covid-19.Thus, the first aim of this repeated cross-sectional study was to compare 24-Hour Movement Guidelines, separately and together, before  and 1-year after the Covid-19 pandemic  in two different subsamples of adolescents. Consistent with previous studies28. During this period , several measures were put in place to prevent the spread of the virus: the movement of people in public spaces was limited between 11:00 PM and 6:00 AM; the capacity of public and/or sporting venues  was limited, playgrounds in the parks were closed, and several federated sports activities were suspended .All data were collected in Extremadura, a region located in southwestern Spain. Both groups were similar in terms of age, sex, and socio-economic status, as all schools included belonged to neighbourhoods with similar socio-demographic characteristicsSocio-demographic data, physical activity, recreational screen time, sleep duration, and academic performance were measured during the two assessments in the two sub-samples.Sociodemographic characteristics. Student’s self-reported age (in years), sex , weight and height. The body mass index (BMI) of each participant was calculated using Cole’s international cut-off points based on their sex and age30.Physical activity. Physical activity was assessed using the Spanish version of a self-reported questionnaire called Physical Activity Questionnaire for Adolescents (PAQ-A) validated by Martínez-Gómez et al.31. This questionnaire has shown to be a valid  and reliable (α = 0.79 and Intraclass Correlation Coefficient [ICC] = 0.71) instrument to assess physical activity levels in Spanish youth aged 12–17 years. In the present study, Cronbach’s alpha of this scale was 0.87. The scale consists of nine items assessing participation in physical activity during the last seven days. Adolescents should respond to the frequency of physical activity participation in a list of activities such as physical education classes, school recess, at lunchtime, right after school, in the evening, and during the last weekend. Each response is scored from 1 to 5 using a Likert-type scale. The average of these scores results in the physical activity index score.Recreational screen time. Recreational screen time was assessed using the Youth Leisure-Time Sedentary Behaviour Questionnaire (YLSBQ) validated by Cabanas-Sánchez et al.32. The questionnaire is a valid (r = 0.36) and reliable (ICC = 0.75) measure to assess sedentary recreational screen time among Spanish young people aged 8 to 18 years32. Students’ self-reported time spent on television, video games, computers, and mobile phones for both weekdays and weekend days. An average of recreational screen time was calculated for each screen-based behaviour in a ratio of 5:2 . The average daily recreational screen time was calculated by summing the different daily screen-based behaviours.Sleep duration. Sleep duration was measured using a Spanish version of Pittsburgh Sleep Quality Index validated in 2003 by Macías and Royuela33. Students self-reported the time they usually go to bed and wake up on both weekdays and weekends. These questions have been shown as a valid (r = 0.45–0.90) and reliable (ICC = 0.71–0.99) measure to assess sleep duration in youths34. The average sleep duration was calculated using the following formula: ([Sleep duration on weekdays × 5] + [Sleep duration on the weekend days × 2]/7).Adherence to the 24-Hour Movement Guidelines. Adherence to the 24-Hour Movement Guidelines established by Tremblay et al.35 was also calculated in this study. Children and youth aged 5–17 years should accumulate at least 60 min of moderate-to-vigorous physical activity per day , spend less than two hours of recreational screen time per day, and sleep between 9 and 11 h per day (children aged 5–13) and 8 to 10 h per day (adolescents aged 14–17) to meet each of the three recommendations35.Academic performance. Academic performance was assessed through school records at the end of the academic year. Academic performance was based on four subjects: first language (Spanish), second language (English), mathematics, and physical education. The grade point average (GPA) was then calculated as an average of the scores in these four subjects. Previous studies have used these subjects as indicators to assess academic performance38.The research team contacted the principal and teachers of the schools to conduct this study. Parents or legal guardians were informed of the purpose of the study by letter prior to data collection, and written informed consent was required from both participants and their parents/legal guardians. Only students who returned written informed consent signed by parents and themselves participated in this study. The paper-and-pencil questionnaire was administered in a regular classroom by one member of the research team to unify the same protocol. The average time taken to complete the set of questionnaires was approximately 20 min.p > 0.01), all analyses were performed on the entire sample.At first step, descriptive statistics were used to examine the average daily time spent in physical activity, recreational screen time, and sleep duration, as well as the adherence to the 24-Hour Movement Guidelines, both separately and for all possible combinations. Sex differences were tested using Student t-test and Chi-squared test for continuous and categorical variables, respectively. A linear regression analysis between 24-Hour Movement Behaviours and interaction sex*predictor was conducted to examine the interaction of sex and each of the movement behaviours . As no significant interaction was found between sex and physical activity, recreational screen time, and sleep duration in relation to academic performance  were estimated. For all models, the estimation of time as within factor (before vs. 1-year after Covid-19 pandemic) and a predictor  were included, as well as the interaction time*predictor. A significant effect indicates that the association between movement behaviours and academic performance was statistically different in terms of time . In all models, age, sex, and BMI were included as covariates. All analyses were performed using SPSS version 23.0 for Windows . The level of significance was set at p < 0.05). In addition, a significantly larger proportion of adolescents, examined before Covid-19 pandemic, met the 24-Hour Movement Guidelines for physical activity, recreational screen time, and sleep duration, independently and all together.Table Table p < 0.001) among adolescents assessed before Covid-19 pandemic. For each one-unit increase in physical activity, the estimation of academic performance would increase by 0.24. However, the association became negative (β = 0.24 + ( − 0.40) =  − 0.26; p < 0.001) among adolescents evaluated 1-year after Covid-19, that is, physical activity was negatively associated with academic performance, and differences in slopes were statistically significant (p < 0.001).Figure p > 0.05) among adolescents assessed before Covid-19 pandemic .Recreational screen time was not associated with academic performance . The interaction effect was not significant (p > 0.05), that is, this association remained significant for adolescents evaluated 1-year after Covid-19 pandemic (β = 0.23 + ( − 0.18) = 0.05; p > 0.05).Lastly, as displayed in Table The first aim of this study was to examine possible changes in 24-Hour Movement Guidelines for adolescents before (T1) and after 1-year (T2) into the Covid-19 pandemic. The second aim examined the possible changes in the relationship between 24-Hour Movement Behaviours and academic performance, before and after 1-year into the Covid-19 pandemic. The main findings of this study are as follows: 1) 24-Hour Movement Behaviours appear to have worsened among young people after 1-year into the Covid-19 pandemic compared to pre-pandemic; 2) adherence to the three 24-Hour Movement Guidelines seem to be significantly lower among Spanish adolescents after 1-year into the Covid-19 pandemic, particularly sleep duration recommendations; 3) the positive relationship between physical activity and academic performance seems to have disappeared 1-year after Covid-19 pandemic; (4) the non-significant relationship between recreational screen time and academic performance and the positive relationship between sleep duration and academic performance does not seem to have changed 1-year after Covid-19.20. However, in contrast to Dubuc's study20, our study also found significantly lower adherence to screen recommendations among young people. The decrease in physical activity and increase in recreational screen time could partly be explained by Covid-19-related restrictions in Spain during second data collection . Although these public health restrictions have been able to reduce the spread of Covid-19, they may also have negatively affected physical activity levels and recreational screen time. Particularly, in this study, sleep recommendations seem to be the most affected 1-year after the Covid-19 pandemic. This could be explained by the increase in recreational screen time, due to the 24-h cycle39. It is also possible that the anxiety and depression caused by Covid-19 may have affected sleep quality, which may have a downstream effect on adolescents' sleep duration41. Therefore, this research suggests that the direct and indirect effects of the Covid-19 pandemic have negatively affected physical activity, recreational screen time, and sleep duration 1-year after into the Covid-19 pandemic.With respect to the first study hypothesis, it was postulated that adherence to 24-Hour Movement Guidelines would be lower in adolescents assessed after 1-year into the Covid-19 pandemic. Before the Covid-19 pandemic, the adherence to the recommendations was 35.2% for physical activity, 13.2% for recreational screen time, and 50.5% for sleep duration among Spanish adolescents. Only 3.3% met all three 24-Hour Movement Guidelines, while 28.9% did not met with any of the three recommendations. These results are worrying because of the low compliance of Spanish adolescents with the recommendations prior to the Covid-19 pandemic. However, these values seem to have worsened 1-year after Covid-19 pandemic among young people, as significant changes were found in physical activity levels , recreational screen time (+ 0.93 h/day) and sleep duration (− 0.56 h/day). Similarly, fewer adolescents seem to meet the recommendations for physical activity (23.4%), recreational screen time (9.2%), and sleep duration (4.0%). Only 0.2% reported meeting all three 24-Hour Movement Guidelines, while 66.3% did not meet any of the three recommendations. The only study published to date that examined changes of 24-Hour Movement Guidelines before and 1-year after into the Covid-19 pandemic is consistent with our results42. However, restrictive measures during Covid-19 have increased stress and anxiety levels43, and limited structured physical activities, which has led to a reduction in physical activity levels, especially in adolescents16. The implementation of these restrictive measures, such as safe distance, use of face mask, avoidance of sharing materials, etc., has encouraged new forms and places to participate in physical activity, such as individual physical activity at home16. In this sense, it is likely that the lack of social interaction and enjoyment of these activities or that they are not performed outdoors could have impacted on the benefits of physical activity on academic performance44. However, since the relationship between physical activity and academic performance 1-year after Covid-19 has not been studied in depth, further mix-method studies examining this relationship are needed.Regarding the second hypothesis, it was postulated that the relationship between 24-Hour Movement Behaviours and academic performance could have changed 1-year after Covid-19 pandemic. The results showed a positive relationship between physical activity and academic performance before the Covid-19 pandemic, whereas this relationship became negative among adolescents evaluated 1-year after Covid-19. The positive effect of physical activity on the brain may be the result of several factors such as increased cerebral blood flow, oxygen to the brain, synaptic plasticity activity, and neurotransmitter secretion levels, resulting in increased levels of arousal, attention, and effort, which have a positive impact on cognitive task performance immediately after physical activity25 found that the amount of time spent using screen-based devices was not associated with academic performance. Specifically, the mentioned systematic review with meta-analysis conducted by Adelantado-Renau et al.25 only found that television viewing and video game use were the only two screen-based devices negatively associated with academic performance. These authors suggest that the type of screen-based devices assessed , the purpose , and the context in which screen media are used  may affect the relationship with academic performance25. The fact that our study also included computer and mobile phone use within the recreational screen time may explain the lack of association between these two variables.Although the amount of recreational screen time appears to have increased after 1-year into the Covid-19 pandemic, there was no change in the relationship between recreational screen time and academic performance before and after 1-year into Covid-19 pandemic, remaining non-significant. These results suggest that recreational screen time does not seem to have affected young people's academic performance before and after 1-year Covid-19. In line with our results, a systematic review with meta-analysis45. In addition, longer sleep duration has been shown to have a positive effect on adolescents’ ability to retain learned information and make it accessible in the long-term memory46. Given that most teachers in Spain use theoretical tests for grading students, it is possible that Spanish adolescents who get enough sleep are better able to retain information more effectively and, consequently, perform better academically47. Therefore, although in the present study sleep duration seems to have decreased after 1-year into the Covid-19 pandemic, it does not seem to have been sufficient to cause changes in its relationship with academic performance.Finally, a positive relationship between sleep duration and academic performance was also found, both before and after 1-year into Covid-19 pandemic. It is important to note that previous studies have shown that both quantity and quality sleep are positively related to cognitive improvements such as better memory, attention, and executive controlThe present study has some limitations that that should be taken into account in future studies. First, this research uses a repeat cross-sectional design in two sub-samples. Although the two sub-samples were similar in terms of age, sex, and socioeconomic status, this design may lead to bias in the results. On the other hand, it does not allow us to examine the directionality of the relationship between 24-Hour Movement Behaviours and academic performance. Future longitudinal studies are needed to reinforce the findings of this study. Secondly, although Covid-19 has affected the entire world population, the restrictions have been different in different countries. Therefore, the results found cannot be extrapolated to the rest of the countries. Thus, more studies that examined changes in 24-Hour Movement Guidelines after 1–2 years into the Covid-19 pandemic are needed. Thirdly, although all questionnaires for measuring 24-Hour Movement Behaviours are valid and reliable, young people may have overestimated or underestimated the time spent on them. Future studies should use device-based measures to assess these three movement behaviours throughout the 24-h period. Fourthly, it was not possible to assess weight and height in the second subsample due to Covid-19 restrictions in the schools evaluated. In this regard, future studies should assess weight and height through electronic scale with a measuring rod. Finally, the use of qualitative methodology could help to explore some of the reasons why adherence to the 24-Hour Movement Guidelines has declined among young people and its relationship with academic performance.Despite these limitations, this study has some strengths. This is one of the first studies that examined 24-Hour Movement Guidelines, separately and together, before and 1-year after the Covid-19 pandemic. In addition, this is the first study that compared the relationship between 24-Hour Movement Behaviours and academic performance before and after Covid-19 pandemic in adolescents. Finally, sex, age, and BMI were introduced as covariates in the analyses to avoid possible bias.The results suggest that physical activity, recreational screen time, and sleep duration appear to have been negatively affected by the Covid-19 pandemic. Particularly, sleep recommendations seem to be the most affected movement behaviour 1-year after the Covid-19 pandemic. Furthermore, 1-year after the Covid-19 pandemic, the relationship between physical activity and academic performance was negative, while recreational screen time and sleep duration with academic performance did not change compared to before Covid-19 results. The reopening of many schools, playgrounds, parks, and organised sports activities in Spain seems to have been insufficient to mitigate the negative consequences of the Covid-19 lockdown on 24-Hour Movement Behaviours. Therefore, there is a serious public health problem that requires immediate and coordinated action by schools, policy makers, health practitioners, and researchers to mitigate the adverse effects of the pandemic on movement behaviours. Specially, it seems very important to design strategies to increase the duration and quality of sleep of young people. Similarly, reducing Covid-related restrictions on physical activity could have a positive impact on academic performance.